A New Proposed Symbiotic Plant–Herbivore Relationship between Burkea africana Trees, Cirina forda Caterpillars and Their Associated Fungi Pleurostomophora richardsiae and Aspergillus nomius

Burkea africana is a tree found in savannah and woodland in southern Africa, as well as northwards into tropical African regions as far as Nigeria and Ethiopia. It is used as fuel wood, medicinally to treat various conditions, such as toothache, headache, migraine, pain, inflammation, and sexually transmitted diseases, such as gonorrhoea, but also an ornamental tree. The current study investigated the possible symbiotic relationship between B. africana trees and the C. forda caterpillars and the mutual role played in ensuring the survival of B. africana trees/seedlings in harsh natural conditions and low-nutrient soils. Deoxyribonucleic acid isolation and sequencing results revealed that the fungal species Pleurostomophora richardsiae was highly predominant in the leaves of B. africana trees and present in the caterpillars. The second most prominent fungal species in the caterpillars was Aspergillus nomius. The latter is known to be related to a Penicillium sp. which was found to be highly prevalent in the soil where B. africana trees grow and is suggested to play a role in enhancing the effective growth of B. africana trees in their natural habitat. To support this, a phylogenetic analysis was conducted, and a tree was constructed, which shows a high percentage similarity between Aspergillus and Penicillium sp. The findings of the study revealed that B. africana trees not only serve as a source of feed for the C. forda caterpillar but benefit from C. forda caterpillars which, after dropping onto the soil, is proposed to inoculate the soil surrounding the trees with the fungus A. nomius which suggests a symbiotic and/or synergistic relationship between B. africana trees and C. forda caterpillars.


Introduction
Burkea africana (Fabaceae: Caesalpinioideae: Caesalpinieae) trees occur in various types of woodland over a wide range of altitudes and habitats but are most characteristic in hot, low-lying areas [1]. The trees are deciduous, and the leaves fall from May to September, and new leaves flush from August to December [2]. Flowers appear from August to November, whereas fruit ripens from February to October and can remain on the tree for a long time [1,3]. In South Africa, B. africana is prevalent in Mpumalanga, Limpopo, and parts of Gauteng Provinces. There have been numerous attempts at growing B. africana trees outside their natural habitat through the excavation of seedlings or the raising of seedlings after the germination of seeds. However, it has been reported that seedlings only survive for 6-8 months after removal from their natural habitat [4]. Burkea africana trees grow in clusters in their natural habitat, also indicating selectivity in conditions to support growth and further development of seedlings [4].
Burkea africana is well known as an important host of the caterpillars of the Caster semi-looper moth Cirina forda (Lepidoptera: Bombycoidea; Saturniidae), with infestation Cirina forda caterpillars are widely used as food in Africa, especially in Nigeria, Zimbabwe, Zambia, South Africa, Central Africa, and the Democratic Republic of Congo [7]. In South Africa, the caterpillars are commonly known as "mashonzha" and are considered a delicacy among the VhaVenda, BaPedi, and VaTsonga, which mainly reside in the Limpopo Province [4]. Recently, C. forda has gained popularity among the AmaNdebele people, which is found mainly in Mpumalanga Province. The larvae are handpicked, squeezed to remove the entrails, boiled in salty water for longer preservation, dried, and sold at the local markets. It is then prepared as a relish served with porridge made of maise meal. Current studies do not report any significant health problems associated with the consumption of edible insects, including C. forda, and are therefore considered safe for consumption. Several studies have been conducted on the nutritional health benefits offered by edible insects indicating that such insects contain sufficient amounts of goodquality protein and other important nutrients [8][9][10]. Cirina forda caterpillars are no exception, known to be high in crude protein and vitamins.
The fungal species, particularly P. richardsiae and A. nomius species, have been reported in the leaves and nuts of different trees. Pleurostomophora richardsiae was initially known as a human pathogen [11][12][13][14] as the cause of subcutaneous phaeohyphomycotic cysts after traumatic implantation [15]. Currently, P. richardsiae, P. repens, Pl. ootheca [16] and P. ochracea [17] are the four species that are recognised. Aspergillus nomius was first described in 1987 [18] and is reported to be the producer of both B and G-type aflatoxins Cirina forda caterpillars are widely used as food in Africa, especially in Nigeria, Zimbabwe, Zambia, South Africa, Central Africa, and the Democratic Republic of Congo [7]. In South Africa, the caterpillars are commonly known as "mashonzha" and are considered a delicacy among the VhaVenda, BaPedi, and VaTsonga, which mainly reside in the Limpopo Province [4]. Recently, C. forda has gained popularity among the AmaNdebele people, which is found mainly in Mpumalanga Province. The larvae are handpicked, squeezed to remove the entrails, boiled in salty water for longer preservation, dried, and sold at the local markets. It is then prepared as a relish served with porridge made of maise meal. Current studies do not report any significant health problems associated with the consumption of edible insects, including C. forda, and are therefore considered safe for consumption. Several studies have been conducted on the nutritional health benefits offered by edible insects indicating that such insects contain sufficient amounts of good-quality protein and other important nutrients [8][9][10]. Cirina forda caterpillars are no exception, known to be high in crude protein and vitamins.
The fungal species, particularly P. richardsiae and A. nomius species, have been reported in the leaves and nuts of different trees. Pleurostomophora richardsiae was initially known as a human pathogen [11][12][13][14] as the cause of subcutaneous phaeohyphomycotic cysts after traumatic implantation [15]. Currently, P. richardsiae, P. repens, Pl. ootheca [16] and P. ochracea [17] are the four species that are recognised. Aspergillus nomius was first described in 1987 [18] and is reported to be the producer of both B and G-type aflatoxins [19]. Aspergillus nomius has been identified in Pistachio nuts [20], wheat [18], maise, and peanuts [19] and in agricultural soils in the US [18,21], Iran [22] and Thailand [23]. No published Information is available regarding the potential role of C. forda in the growth and development of B. africana trees. Although several studies have been conducted on the medicinal properties of B. africana, to date, no information is available on why the caterpillars are selectively attracted to B. africana trees to feed on the leaves. A previous study clearly showed the differences in the soil around the trees (Burkea soils) in comparison to other soils (non-Burkea soils), using soil metabolomic analysis. The nutrient content, plant growth regulating compounds, as well as microorganism differentiation was described by Nemadodzi et al. [4]. The aim of the current study was to elucidate the possible symbiotic relationship that exists between the caterpillars and the trees and their potential role in the growth and establishment of B. africana trees in their natural environment to explain and support the soil metabolomics analysis as reported by Nemadodzi et al. [4]. This is the first study to report on the presence of two fungal species found in or on the leaves of B. africana trees and C. forda caterpillars ( Figure 1) and the suggested co-dependent symbiotic relationship between the caterpillars and the trees.

Collection of Leaf and Caterpillar Sample
Newly developed leaves were harvested in October 2017 from randomly selected B. africana trees at Telperion Game Reserve, which covers approximately 1000 ha and is situated in Mpumalanga province, South Africa. Cirina forda caterpillars were randomly collected from these trees by handpicking them from the leaves and the ground surrounding B. africana trees in November 2017. The study was conducted at three different sites within the reserve, namely site 1 (S 25 •  . Telperion is situated in the summer rainfall region of South Africa, with annual rainfall ranging from 570-730 mm [24]. According to Brown et al. [25], the average temperature indicates February to be the hottest month of the year, with 26.4 • C as the average daily maximum, whilst 15.1 • C is the average daily minimum. The collection period of September-November falls within the spring and summer seasons, with temperatures ranging from a maximum of 24 • C and a minimum of 12 • C.

Genomic DNA PCR and Sequencing
Fresh leaves of B. africana were harvested at Telperion Game Reserve and placed in brown bags, and stored at −80 • C until use. A total of 30 live caterpillars were handpicked, and the intestines were squeezed out and put in enclosed bottles which were stored at −80 • C to limit microbial contamination. Both the leaves and caterpillars were sent to Inqaba Biotechnical Industries, a commercial service provider, for next-generating sequencing (NGS) for the identification of differences in a mixed microbial species [26] and/or population in a sample through purifying and sequencing following the protocol below: ITS Metagenomics: (V3) regions were amplified in a 25 uL reaction using Q5 ® Hot start High-Fidelity 2× Master Mix (New England Biolabs, Ipswich, MA, USA). Amplicon library PCR was performed on all replicate extractions separately. The DNA primers used were Truseq-tailed ITS 1F and ITS 4. Thermocycler settings for PCR amplification were as follows: (1) initial denaturation at 95 • C for 2 min (2) 30 cycles of 95 • C for 20 s . The final pooled library was at 10 pM with 20% PhiX as control. 20 Mb of data of 2 × 300 bp long reads per sample were produced. The list of primers and sequences used for the detection of fungal species is provided in Table 1. Table 1. List of primers used.

Soil Analysis
Soil samples were collected at the same study sites at Telperion Game Reserve as the leaves and caterpillars samples. Soil analysis was performed on 30 samples of Burkea soils representing the rhizosphere and another 30 samples of non-Burkea soils representing the non-rhizosphere soils. Soil samples (500 mg) were subjected to DNA extraction using a NucleoSpin Soil DNA kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions, and results were confirmed with agarose gel before sending for Polymerase Chain Reaction (amplification and cloning of DNA) and sequencing at Inqaba Biotechnology industry, Pretoria, South Africa as previously described [4].

Higher Order Classification of the Microorganisms in the Caterpillars
The results of the study showed that the Fungal kingdom was most prevalent (96.78%), followed by an uninformative Kingdom (3.17%), which could not be classified and/or accurately identified under any kingdom. Bacteria and Plantae had the same percentages of 0.03 whilst Protozoa occupied the least percentages of 0.01, respectively, which yielded poor sequences; therefore, these were not accessioned or subjected to further analysis.

Soil Analysis
Soil samples were collected at the same study sites at Telperion Game Reserve as the leaves and caterpillars samples. Soil analysis was performed on 30 samples of Burkea soils representing the rhizosphere and another 30 samples of non-Burkea soils representing the non-rhizosphere soils. Soil samples (500 mg) were subjected to DNA extraction using a NucleoSpin Soil DNA kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions, and results were confirmed with agarose gel before sending for Polymerase Chain Reaction (amplification and cloning of DNA) and sequencing at Inqaba Biotechnology industry, Pretoria, South Africa as previously described [4].

Higher Order Classification of the Microorganisms in the Caterpillars
The results of the study showed that the Fungal kingdom was most prevalent (96.78%), followed by an uninformative Kingdom (3.17%), which could not be classified and/or accurately identified under any kingdom. Bacteria and Plantae had the same percentages of 0.03 whilst Protozoa occupied the least percentages of 0.01, respectively, which yielded poor sequences; therefore, these were not accessioned or subjected to further analysis.

Family Classification of Ascomycota in the Caterpillars
Pleurostomophora dominated (60.08%), followed by Trichocomaceae (32.91%). The third and fourth families were uninformative and could not be assigned to any classification and were recorded at 6.08 and 0.45%, respectively.

Species Classification
The species which took predominance was the fungi Pleurostomophora richardsiae (60%); the second dominant was Aspergillus nomius (32%) (Figure 3). Pleurostomophora dominated (60.08%), followed by Trichocomaceae (32.91%). The third and fourth families were uninformative and could not be assigned to any classification and were recorded at 6.08 and 0.45%, respectively.

Species Classification
The species which took predominance was the fungi Pleurostomophora richardsiae (60%); the second dominant was Aspergillus nomius (32%) (Figure 3). Each fungal species detected and identified in C. forda was represented by a specific accession number and confirmed by NIH in the National Library Medicine at the National Centre for Biotechnology Information (Table 2).  Each fungal species detected and identified in C. forda was represented by a specific accession number and confirmed by NIH in the National Library Medicine at the National Centre for Biotechnology Information (Table 2).
An operational taxonomy unit (OTU) was done to indicate clustering and long reads to generate percentage identity of the species identified in C. forda caterpillars, produce more accurate and prediction fungal species as shown in Table 3.  The results of tree construction, replication and scale used in a phylogenetic tree indicating the probability and higher percentages of mean close relatedness of fungal species are Figure 4.

Classification of the Microorganisms in the Leaves
The results of the study showed that the Fungal kingdom was most p (99.47%), followed by an uninformative Kingdom (0.46%), which could not be c

Classification of the Microorganisms in the Leaves
The results of the study showed that the Fungal kingdom was most prevalent (99.47%), followed by an uninformative Kingdom (0.46%), which could not be classified under any kingdom, and Protozoa had the lowest percentage of 0.7, which yielded low similarity; therefore, these were not accessioned or subjected to further analysis.

Phylum Classification
The leaves of B. africana were dominated by fungi, notably Ascomycota (94%), followed by an unknown phylum (5%), with other phyla, such as Tracheophyta, Proteobacteria and Ciliophora, at almost undetectable levels.

Family Classification of the Ascomycota in the Leaves
Pleurostomophora (72%) was found to be the most prevalent family, followed by Togniniaceae (14%) and Polyporaceae (6.05%).

Species Classification
The species which took predominance was the fungi Pleurostomophora richardsiae (72%); the second dominant was Phaeoacremonium scolyti (14%), as demonstrated in Figure 5. The leaves of B. africana were dominated by fungi, notably Ascomycota (94%), followed by an unknown phylum (5%), with other phyla, such as Tracheophyta, Proteobacteria and Ciliophora, at almost undetectable levels.

Family Classification of the Ascomycota in the Leaves
Pleurostomophora (72%) was found to be the most prevalent family, followed by Togniniaceae (14%) and Polyporaceae (6.05%).

Species Classification
The species which took predominance was the fungi Pleurostomophora richardsiae (72%); the second dominant was Phaeoacremonium scolyti (14%), as demonstrated in Figure 5. Each fungal species detected and identified in the leaves of B. africana was represented by a specific accession number and confirmed by NIH in the National Library of Medicine of the National Centre for Biotechnology Information (see Table 4).  Each fungal species detected and identified in the leaves of B. africana was represented by a specific accession number and confirmed by NIH in the National Library of Medicine of the National Centre for Biotechnology Information (see Table 4).  An operational taxonomy unit (OTU) was done to indicate clustering and long reads to generate percentage identity of the species identified in B. africana leaves, produce more accurate and prediction fungal species as shown in Table 5.  The results of the tree construction, replication and scale used in a phylogenetic tree indicating the probability and higher percentages of mean close relatedness of fungal species are shown Figure 6 below.

Discussion
The two fungal species, P. richardsiae and A. nomius were identified with high prevalence from the C. forda caterpillars, which were collected from B. africana trees. Additionally, P. richardsiae was dominant in both the C. forda caterpillars (60%) and the leaves (72%) of B. africana trees, as shown in Figures 3 and 5, respectively. Findings from a previous study reported that Penicillium sp. was the most prevalent fungal species in the Burkea soils, whereas it was absent in the non-Burkea soils, indicative of the important role of the fungal species in providing a supportive soil environment for the trees to survive [4]. The BLAST analysis could not identify all the Kingdom, Family, and fungal species accurately, resulting in uninformative classification and Protozoa detection. Protozoa were detected in the BLAST analysis, although the BLAST analysis was not using a prokaryote-specific database.
Carlucci et al. [27] showed that Penicillium sp. could be divided into two subgenera (Penicillium and Aspergilloides). Penicillium and Aspergillus are therefore regarded as sister genera due to sharing of a common ancestor and microbial divergence [28]. Similar findings were confirmed by Crous et al. [29]. The International Commission on Penicillium and Aspergillus (ICPA) met in Utrecht, the Netherlands, and discussed the implications of the single-name nomenclature on Aspergillus and Penicillium taxonomy [30]. The similarities have often been described between members of the two genera. Carlucci et al., Houbraken et al., Visagie et al. [27,31,32] reported that Aspergillus paradoxus produce conidial heads with a terminal vesicle reminiscent of Aspergillus yet belong to Penicillium subgenus Penicillium.
It was, however, expected to find A. nomius together with Penicillium in the soil, for them to act as growth-inoculant fungi. The collection of samples from the soil and caterpillars from different locations and in different years might therefore explain why different strains of the same fungus were collected, and therefore, it is put forward that Aspergillus nomius and Penicillium are related fungi with slight differences in genetic makeup as reported by Crous et al. [29]. The presence of A. nomius species detected in the caterpillars hosted by B. africana trees and Penicillium in the Burkea-soil (soil where B. africana trees grow), therefore, confirms the link between the caterpillars as a host of Aspergillus/Penicillium sp. fungus. It is therefore proposed that the caterpillar is the source of inoculation of A. nomius/Penicillium sp., which serves as a constant and continuous source of inoculum in the soil. [4]. Since the similarities in the two fungal species have been reported in previous research, the current results confirm that Aspergillus and Penicillium are sister genera/fungal species, as evidently shown in the phylogenetic tree constructed (see Figure 6).
This study reported the presence of various growth-promoting metabolites (GPM) in Burkea-soils due to the presence of Aspergillus/Penicillium sp., where the fungal composition was linked to the development of B. africana trees and is assumed to be responsible for creating a supportive environment for the natural establishment and survival of seedlings [4]. The abovementioned is based on the absence of a dominant fungal species never found in any of the non-Burkea soils, even though the soil collections were often performed a few meters apart. The presence of a dominant fungal species in all the Burkea soils is therefore important, as it necessitates a continuous inoculum of the soil, especially around the trees.
Pleurostomophora richardsiae is an emergent fungal pathogen that has been associated with esca and Petri disease in California [33] and caused vascular discolouration after field and glasshouse inoculations similar to that seen in Petri-diseased grapevines in South Africa [34]. It is a rare dematiaceous (dark-walled) fungus that was previously known as Phialophora richardsiae but has been recently renamed [35]. It was first isolated from a patient with a phaeomycotic cyst in 1968 [36] and is found in the soil, decaying wood and vegetation [37]. Levenstadt et al. [38] reported that P. richardsiae was dominant in the leaves of almond trees. It is also considered the most aggressive pathogen among several other fungi found in almond trees [38], and its aggressiveness may be related to the concentration level found in the leaves, which in turn causes severe mechanical damage during and after the caterpillars' invasion. In the current study, P. richardsiae fungus was also found to be highly dominant in the caterpillars, as shown in Figure 3.
The current study represents the first report of P. richardsiae to be the main fungal species in or on the leaves of B. africana trees. This is also the first study to report that P. richardsiae is also found to be prevalent in the C. forda caterpillars, which feed on the leaves of B. africana trees. The fungal species is, however, not a deadly pathogen to the tree, as there are no reports of the death of B. africana trees caused by an infestation by C. forda caterpillars, although they cause severe defoliation by feeding on the leaves.
It is therefore suggested that P. richardsiae lives inside or outside on the B. africana leaves, and it is proposed that it indirectly influences host location and oviposition behaviour of Castor semi-looper moths which lays eggs on the leaves/branches of B. africana trees, which later hatch into C. forda caterpillars. This was also reported by Olmo et al., Vannette et al., Ballhorn et al., who stated that fungi are known to be important mediators of plant-herbivore interactions.
Furthermore, other studies conducted by Locke and Crawford, Fontaine et al. [43,44] suggested that P. richardsiae is involved in the release of plant volatiles. It is, therefore, also possible that P. richardsiae plays a major role in attracting Castor semi-looper moths by releasing plant volatiles as cues when searching for their host to lay the eggs on, as the start of a life cycle of the caterpillar recorded from November 2021-January 2022 as demonstrated in Figure 7. The results of the study suggest that P. richardsiae plays a mediating role in B. africana-moth/caterpillar interactions.
other fungi found in almond trees [38], and its aggressiveness may be related to the concentration level found in the leaves, which in turn causes severe mechanical damage during and after the caterpillars' invasion. In the current study, P. richardsiae fungus was also found to be highly dominant in the caterpillars, as shown in Figure 3.
The current study represents the first report of P. richardsiae to be the main fungal species in or on the leaves of B. africana trees. This is also the first study to report that P. richardsiae is also found to be prevalent in the C. forda caterpillars, which feed on the leaves of B. africana trees. The fungal species is, however, not a deadly pathogen to the tree, as there are no reports of the death of B. africana trees caused by an infestation by C. forda caterpillars, although they cause severe defoliation by feeding on the leaves.
It is therefore suggested that P. richardsiae lives inside or outside on the B. africana leaves, and it is proposed that it indirectly influences host location and oviposition behaviour of Castor semi-looper moths which lays eggs on the leaves/branches of B. africana trees, which later hatch into C. forda caterpillars. This was also reported by Olmo et al., Vannette et al.,Ballhorn et al., who stated that fungi are known to be important mediators of plant-herbivore interactions.
Furthermore, other studies conducted by Locke and Crawford, Fontaine et al. [43,44] suggested that P. richardsiae is involved in the release of plant volatiles. It is, therefore, also possible that P. richardsiae plays a major role in attracting Castor semi-looper moths by releasing plant volatiles as cues when searching for their host to lay the eggs on, as the start of a life cycle of the caterpillar recorded from November 2021-January 2022 as demonstrated in Figure 7. The results of the study suggest that P. richardsiae plays a mediating role in B. africana-moth/caterpillar interactions. These caterpillars are collected fresh, killed, preserved by adding salt and dried, thereafter sold in the streets markets by street vendors, who are mostly women. The caterpillars are considered a delicacy, eaten as a side dish (after they are boiled and fried) with pap, normally known as vhuswa, which is a hard porridge made of ground maise. Cirina forda caterpillars are known to be a high source of protein [45]. These caterpillars are collected fresh, killed, preserved by adding salt and dried, thereafter sold in the streets markets by street vendors, who are mostly women. The caterpillars are considered a delicacy, eaten as a side dish (after they are boiled and fried) with pap, normally known as vhuswa, which is a hard porridge made of ground maise. Cirina forda caterpillars are known to be a high source of protein [45].
The processing of the caterpillars has been shown to introduce significant changes in some of the nutrients. Decreases in the concentration of nutrients, such as sodium, potassium, iron, magnesium, zinc and copper, were found in processed caterpillars as compared to fresh caterpillars [7].
Aspergillus nomius is a ubiquitous group of filamentous fungi spanning over 200 million years of evolution [46][47][48][49][50][51]. Aspergillus nomius is an aflatoxin-producing member of Aspergillus section Flavi that shows a cosmopolitan distribution. It has been described so far as a human pathogen in a case of breakthrough pneumonia in a patient with acute myeloid leukaemia [52]. In parallel, A. nomius has also been isolated from single cases of keratitis after ocular injury and onychomycosis in otherwise healthy patients [53,54]. Among the over 185 aspergilli, there are several that have an impact on human health and society [53], including 20 human pathogens, as well as beneficial species used to produce foodstuffs and industrial enzymes [55][56][57].
Furthermore, A. nomius is exceptional among microorganisms in being both a primary and opportunistic pathogen, as well as a major allergen [58][59][60][61][62][63]. This is supported by the relationship between C. forda and B. africana trees, which has shown that the infestation intensity does not result in the death of B. africana trees, except for severe defoliation. Aspergillus nomius produce carcinogenic secondary metabolites known as aflatoxins [64][65][66][67][68] responsible for hepatotoxic and immunosuppressive properties in humans and other animals [68,69] which may render agricultural products unusable as feeds and can lead to significant economic loss [70]. Several human case of ocular infection by A. nomius also has been documented [71][72][73] and several aflatoxin outbreaks in humans, following consumption of contaminated grain, have been documented [74][75][76][77]. Its conidia production is prolific and so human respiratory tract exposure is almost constant [78,79]. Concurrently, Aspergillus in human CARD9 deficiency has been referred as a fungal agent that shows predilection for non-pulmonary sites with little impact on the lungs [52]. Aspergillus nomius has been reported from tree nuts [80][81][82][83][84] sugarcane [85][86][87][88] and on assortment of seeds and grain [89][90][91][92]. Originally, A. nomius was considered rare, however, numerous studies have indicated that A. nomius is widely distributed and might be of economic importance [93].
Aspergillus nomius is often associated with insects, such as alkali bees [89] and Formosan subterranean termites [78] and is frequently isolated from insects' frass in silkwormrearing houses in Eastern Asia, Japan, and Indonesia. [56,80,[94][95][96] also reported that A. nomius is found in dead or diseased insects.
Crops infected by A. nomius are the main sources for establishing soil populations, especially when colonized plant material is deposited onto the soil [97]. It is suggested that dead caterpillar bodies which fails to pupate and are found scattered around B. africana trees as shown in Figure 8, could serve as soil inoculum of Penicillium sp. which was found to be highly dominant in the soil where B. africana grows successfully [4]. Figure 9 illustrate the plant-herbivore and fungal species interaction for the effective growth of B. africana trees.
In addition, the current findings further suggest that large amounts of frass/droppings which are excreted by the caterpillars after feeding on the leaves onto the soils surrounding B. africana trees which ultimately, decompose and later inoculate the soil, could likely be involved in enhancing the growth of B. africana seedlings, although further research is needed to confirm this. What could be seen as an attack through the infestation of B. africana trees by C. forda caterpillars, supposedly colonize the caterpillars with A. nomius and later fall to the ground, decay, and in the process becomes a primary inoculum in the soils where B. africana trees grows. In the absence of a continuous introduction of inoculum into the soil by C. forda caterpillars, the fungal species are probably not maintained in the soil and might explain why tree and seedling growth outside its natural environments have not been successful. The absence of C. forda caterpillars found to be highly prevalent with A. Nomius reported to be related to Penicillium sp. could mean different soil composition which will not be conducive and favourable for continuous growth of B. africana, thus ultimately causing a slow death of B. africana seedlings grown outside their natural environment as reported by Nemadodzi et al. [4]. The factors which contribute and influence the release of volatile compounds which serve as an attractant of Castor-semi looper moths to lay their eggs on B. africana trees, however, is still not known which calls for further research.   In addition, the current findings further suggest that large amounts of frass/droppings which are excreted by the caterpillars after feeding on the leaves onto the soils surrounding B. africana trees which ultimately, decompose and later inoculate the soil, could Processing of the caterpillars before consumption forms part of indigenous knowledge, to probably remove most of the harmful contents from the caterpillars before consumed, although information on the removal of fungal species in processing has not been investigated yet. Preparation of the caterpillars before consumption, includes removal of the intestines, and the caterpillars are boiled, dried, and fried before eating. This might also explain that no adverse effects have been reported by consumers of these caterpillars, although P. richardsiae and A. nomius are present in these caterpillars that are consumed. This however warrant future research to determine the role of processing of the caterpillars in reducing or even eliminating the fungal species before consumption.

Conclusions
Growing B. africana trees outside their natural habitat have proven difficult, which is the main reason these trees are not found in nurseries and not commercialised although highly regarded as an ornamental tree. Based on the findings of the current study, it is suggested that two fungal species play an important and integral role in plant-herbivore interactions to ensure the survival of the tree in harsh and challenging environmental conditions. Pleurostomophora richardsiae which is present in the leaves and the intestines of the caterpillars, provides a link to the association of the caterpillars with B. africana trees. A. nomius (reported to be related to Penicillium found in Burkea-soil) found in the C. forda caterpillars, which invade B. africana trees is hypothesised to play a substantial role in the growth and establishment of B. africana trees by being the main, continuous, and primary soil inoculant through colonization of their dead bodies which ultimately plays a vital role of enhancing and influencing the growth of B. africana trees and seedlings. This further reveals the mutual relationship which exists between C. forda caterpillars and B. africana trees as a host and source of food with C. forda playing a role as primary soil inoculants. Future research should be conducted to confirm and identify the possibility of volatile organic compounds which are released from trees that serve as cues in attracting the Castor-semi looper moths. Both the fungal species P. richardsiae and A. nomius present in the caterpillars have been previously recorded as human pathogens. This might raise a concern regarding the consumers and future studies should demonstrate the effect of these fungi on the larval consumer population. The traditional preparation and processing methods might be removing most of the pathogens and lower the risk of pathogen intake, although this warrants further research. Author Contributions: Conceptualization, G.P.; methodology, G.P., software, G.P. and L.E.N.; validation, G.P.; formal analysis, G.P. and L.E.N.; investigation, L.E.N.; resources, G.P., data curation, G.P.; writing-original draft preparation, L.E.N.; writing-review and editing, L.E.N. and G.P.; visualization, L.E.N. and G.P.; supervision, G.P.; project administration, G.P.; funding acquisition, L.E.N. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: Data will be made available upon request.